Antimicrobial and radioprotective compounds

ABSTRACT

The present invention relates to a method of treatment and/or prophylaxis of a microbial infection, comprising administering an effective amount of a compound of formula I as defined herein, or pharmaceutically acceptable salts or derivatives, pro-drugs, tautomers and/or isomers thereof. The invention also relates to a method for protecting a subject from radiation damage, a method of cancer radiotherapy and use as an antimicrobial or radioprotective agent of the compound of formula I. Some of the compounds of formula I are novel and are also described in the present invention, together with pharmaceutical or veterinary compositions containing them.

[0001] This invention relates to compounds which have antimicrobial andradioprotective activity. In particular, the invention relates tosubstituted nitrostyrene compounds which have activity against a widespectrum of organisms including bacteria, fungi and protozoa. Thecompounds of the invention also have the ability to provide protectionfrom radiation damage.

BACKGROUND OF THE INVENTION

[0002] All references, including any patents or patent applications,cited in this specification are hereby incorporated by reference. Noadmission is made that any reference constitutes prior art. Thediscussion of the references states what their authors assert, and theapplicants reserve the right to challenge the accuracy and pertinency ofthe cited documents. It will be clearly understood that, although anumber of prior art publications are referred to herein, this referencedoes not constitute an admission that any of these documents forms partof the common general knowledge in the art, in Australia or in any othercountry.

[0003] Bacterial, fungal and protozoal pathogens are responsible for avery wide variety of infections, ranging from minor respiratory ailmentsto fulminant systemic infections and chronic illnesses. Food poisoningcaused by organisms such as Salmonella or Campylobacter is common, andis often associated with endemic infection in livestock or poultryraised using intensive animal husbandry techniques.

[0004] Despite the wide availability of antibiotics, control ofinfection is difficult, and many organisms have the ability to developresistance. Many microorganisms cause problems which have hithertoproved to be quite intractable, such as multi-drug resistantStaphylococcus aureus infection in hospitals, drug-resistantEnterococcus infections, bacterial, fungal and protozoal infection inHIV patients, tuberculosis, and malaria and other endemic infections inunderdeveloped countries.

[0005] Currently there are only very few agents which have a widespectrum of activity against pathogens of bacterial, fungal andprotozoal origin. Antibiotics are the most widely used agents in thefight against pathogenic microorganisms. However, most antibiotics havenarrow specificity. Even broad spectrum antibacterial antibiotics arenot very effective against fungi and protozoa. Most antibiotics belongto a restricted range of classes of compounds; although improvedsemi-synthetic derivatives of these have developed, only a few newantibiotic compound classes have become available in the last twentyyears.

[0006] The choice of agents for protection of living organisms againstradioactive radiation is also quite limited. Among the radiationprotectors the most effective are sulphur-containing compounds (Kuna,1989). For example, cystamine is approved for use as aradiation-protective agent (Vladimirov et al, 1989). The index ofprotection of this preparation does not exceed 1.45, and has thedisadvantage that it causes diarrhoea. Another knownradiation-protective preparation is mercamine (β-mercaptoethylamine)(Mashkovskiy, 1986).

[0007] This has a low therapeutic index, short period of action (0.5-1h), and short duration of radiation protecting activity (15-30 min).

[0008] It is known that β-nitrostyrene and some of its derivativesdemonstrate biological, and partly fungicidal activity (Foyer, 1973).Russian Patent No. 2145215 showed that certain derivatives ofarylnitroalkenes have antimicrobial, antifungal, antiprotozoal activity,and are able to provide protection from radiation damage. Thesecompounds have the following formula

[0009] in which R₁′ is H or CH₃; and

[0010] R₂′ and R₃′ are the same or different and are selected from H,OCH₃, OH, NO₂ and (CH₃)₂N.

[0011] The activities of these compounds are satisfactory, but there isa need for low cost, low-toxicity agents with a wide spectrum ofantimicrobial activities.

[0012] We have now found that certain substituted nitrostyrene compoundshave excellent activity against very wide spectrum of organisms,including bacteria, fungi and protozoa and also have the ability toprovide protection from radiation damage.

SUMMARY OF THE INVENTION

[0013] The invention provides a method of treatment and/or prophylaxisof a microbial infection, comprising the step of administering aneffective amount of a compound of formula I:

[0014] in which

[0015] X and Y are either the same or different and selected from aheteroatom;

[0016]

is a double or single bond depending on the heteroatoms X and Y;

[0017] R₁ to R₅ are either the same or different and selected fromhydrogen or a non-deleterious substituent; and

[0018] R₆ and R₇ are either the same or different and selected fromhydrogen and a non-deleterious substituent or one of R₆ and R₇ areabsent when there is a double bond present,

[0019] pharmaceutically acceptable salts or derivatives, pro-drugs,tautomers and/or isomers thereof.

[0020] The invention also provides use of the compound of formula I inthe manufacture of a medicament for the treatment and/or prophylaxis ofa microbial infection.

[0021] The invention further provides use of the compound of formula Ifor the treatment and/or prophylaxis of a microbial infection.

[0022] The invention still further provides a method for protecting asubject from radiation damage which comprises administering an effectiveamount of the compound of formula I to a subject in need thereof.

[0023] In another aspect, the invention provides a method of cancerradiotherapy which comprises administering to a subject in need of suchtherapy an effective amount of the compound of formula I and subjectingthe locus of a tumour in the subject to a radiation source.

[0024] In a further aspect, the invention provides use of the compoundof formula I as an antimicrobial or radioprotective agent.

[0025] Preferably X and Y are either the same or different and selectedfrom O and N, more preferably both X and Y are oxygen.

[0026] Preferably R₁ and R₂ are either the same or different andselected from hydrogen, hydroxy, halogen or optionally substituted C₁₋₆alkyl.

[0027] R₃ to R₅ are preferably either the same or different and selectedfrom hydrogen, hydroxy, halogen, nitro, C₁₋₆ alkoxy or optionallysubstituted C₁₋₆ alkyl.

[0028] Preferably halogen is chlorine or bromine.

[0029] The E isomer of the compounds of formula I is preferred.

[0030] Particularly preferred are compounds of the formula I in which X,Y,

, R₆ and R₇ are as defined above; R₁ and R₂ are either the same ordifferent and selected from hydrogen, hydroxy, Cl, Br and C₁₋₄ alkyl;and R₃ to R₅ are either the same or different and selected fromhydrogen, hydroxy, Cl, Br, nitro, C₁₋₄ alkoxy or C₁₋₄ alkyl.

[0031] Specific examples of the compounds of the present invention areas follows:

[0032] (1) X and Y are O, R₁ is methyl and R₂ and R₃ are hydrogen(3,4-methylenedioxy-β-methyl-β-nitrostyrene)

[0033] (2) X and Y are O and R₁ to R₃ are hydrogen(3,4-methylenedioxy-β-nitrostyrene)

[0034] (3) X is N, Y is NH, R₁ is methyl and R₂ and R₃ are hydrogen(benzimidazole-5-β-nitropropylene)

[0035] (4) X is N, Y is NH, R₁ is hydrogen, R₂ is methyl and R₃ isabsent (2-methyl benzimidazole-5-β-nitroethylene)

[0036] (5) X is O, Y is N, R₁ and R₂ are hydrogen and R₃ is absent(benzoxazole-5-β-nitroethylene)

[0037] (6) X is N, Y is O, R₁ and R₂ are methyl and R₃ is absent(2-methyl benzoxazole-5-β-nitropropylene)

[0038] Some of the compounds of the formula I are novel per se.

[0039] Accordingly, the invention provides a compound of formula Ia:

[0040] in which X, Y,

and R₁ to R₇ are as defined in formula I above,

[0041] with the provisos that

[0042] (i) when both X and Y are O and R₂ to R₇ are hydrogen, then R₁ isnot hydrogen, C₁₋₄ alkyl, CO₂Et,

[0043] or CH₂CO₂R₈ in which R₈ is C₁₋₁₂ alkyl or phenyl optionallysubstituted by one or more halogen;

[0044] (ii) when both X and Y are O, then R₁ to R₇ are not hydrogen;

[0045] (iii) when R₂, R₆ and R₇ are hydrogen, one of R₃, R₄ or R₅ ismethyl and X and Y are O, then R₁ is not methyl;

[0046] (iv) when R₂, R₃, R₅, R₆ and R₇ are hydrogen, R₄ is OCH₃ and Xand Y are O, then R₁ is not H, CH₃ or CH₂CH₃;

[0047] (v) when R₁ to R₅ are H and X and Y are O, then at least one ofR₆ and R₇ is not methyl;

[0048] (vi) when R₂, R₄, R₅, R₆ and R₇ are H, R₃ is OCH₃ and X and Y areO, then R₁ is not CH₃; and

[0049] (vii) when R₁, R₂, R₃, R₅, R₆ and R₇ are H and X and Y are O,then R₄ is not OCH₃.

[0050] The invention also provides a process for the preparation of thecompound of formula Ia defined above which comprises condensing acompound of formula II:

[0051] in which X, Y,

, R₃ to R₇ are as defined in formula Ia above

[0052] with a compound of formula III:

R₁R₂CHNO₂  III

[0053] in which R₁ and R₂ are as defined in formula Ia above.

[0054] The invention further provides a process for the preparation ofthe compound of formula Ia defined above which comprises reacting acompound of formula IV:

[0055] in which X, Y,

, R₁ to R₇ are as defined in formula Ia above with C(NO₃)₄.

[0056] The processes are preferably performed in the presence of acatalyst, such as, an amine or an alkali metal hydroxide, for example,NaOH or KOH.

[0057] In a further aspect, the invention provides a pharmaceutical orveterinary composition comprising the compound of formula Ia definedabove together with a pharmaceutically or veterinarily acceptablecarrier.

[0058] Preferably, the pharmaceutical or veterinary composition is atopical, oral or parenteral composition.

[0059] The pharmaceutically or veterinarily acceptable carrier ispreferably an organic solvent such as acetone, benzene, acetonitrile,DMSO or an alcohol, for example, methanol or ethanol. While thecompounds of the present invention show a poor solubility in water, whenwater is combined with an organic solvent a stable mixture is formed.

DETAILED DESCRIPTION OF THE INVENTION

[0060] For the purposes of this specification it will be clearlyunderstood that the word “comprising” means “including but not limitedto”, and that the word “comprises” has a corresponding meaning.

[0061] The term “heteroatom” denotes O, N or S.

[0062] The term “non-deleterious substituent” is used herein in itsbroadest sense and refers to a substituent which does not have adeleterious effect on the antimicrobial or radioprotective properties ofthe compound. Examples include alkyl, alkenyl, alkynyl, aryl, halo,haloalkyl, haloalkenyl, haloalkynyl, haloaryl, hydroxy, alkoxy,alkenyloxy, aryloxy, benzyloxy, haloalkoxy, haloalkenyloxy, haloaryloxy,nitro, nitroalkyl, nitroalkenyl, nitroalkynyl, nitroaryl,nitroheterocyclyl, amino, alkylamino, dialkylamino, alkenylamino,alkynylamino, arylamino, diarylamino, benzylamino, dibenzylamino, acyl,alkenylacyl, alkynylacyl, arylacyl, acylamino, diacylamino, acyloxy,alkylsulphonyloxy, arylsulphenyloxy, heterocyclyl, heterocycloxy,heterocyclamino, haloheterocyclyl, alkylsulphenyl, arylsulphenyl,carboalkoxy, carboaryloxy mercapto, alkylthio, arylthio, acylthio andphosphorus-containing compounds.

[0063] Particularly suitable non-deleterious substituents are alkyl,alkenyl, alkynyl, halo, haloalkyl, haloalkenyl, haloalkynyl, hydroxy,alkoxy, alkenyloxy, haloalkoxy, haloalkenyloxy, nitro, nitroalkyl,nitroalkenyl and nitroalkynyl.

[0064] In a preferred embodiment the non-deleterious substituents areC₁₋₆ alkyl, halo, hydroxy, C₁₋₆ alkoxy and nitro.

[0065] The term “optionally substituted” means that a group may or maynot be further substituted with, for example, the groups specified aboveunder the definition of non-deleterious substituent.

[0066] The term “halogen” refers to fluorine, chlorine, bromine andiodine, preferably chlorine and bromine.

[0067] The term “alkoxy” is used herein in its broadest sense and refersto straight chain, branched chain or cyclic oxy-containing radicals eachhaving alkyl portions, preferably C₁₋₆ alkyl, more preferably C₁₋₄alkyl. Examples of such alkoxy groups are methoxy, ethoxy, propoxy,butoxy and t-butoxy.

[0068] The terms “C₁₋₄ alkyl” or “C₁₋₆ alkyl” used either alone or incompound words such as “optionally substituted C₁₋₄ or C₁₋₆ alkyl” referto straight chain, branched chain or cyclic hydrocarbon groups havingfrom 1 to 6 carbon atoms. Illustrative of such alkyl groups are methyl,ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl,pentyl, neopentyl, hexyl, cyclopropyl, cyclobutyl, cyclopentyl orcyclohexyl.

[0069] The salts of the compound of formula I or Ia are preferablypharmaceutically acceptable, but it will be appreciated thatnon-pharmaceutically acceptable salts also fall within the scope of thepresent invention, since these are useful as intermediates in thepreparation of pharmaceutically acceptable salts. Examples ofpharmaceutically acceptable salts include salts of pharmaceuticallyacceptable cations such as sodium, potassium, lithium, calcium,magnesium, ammonium and alkylammonium; acid addition salts ofpharmaceutically acceptable inorganic acids such as hydrochloric,orthophosphoric, sulphuric, phosphoric, nitric, carbonic, boric,sulfamic and hydrobromic acids; or salts of pharmaceutically acceptableorganic acids such as acetic, propionic, butyric, tartaric, maleic,hydroxymaleic, fumaric, citric, lactic, mucic, gluconic, benzoic,succinic, oxalic, phenylacetic, methanesulphonic,trihalomethanesulphonic, toluenesulphonic, benzenesulphonic, salicylic,sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic,lauric, pantothenic, tannic, ascorbic and valeric acids.

[0070] In addition, some of the compounds of the present invention mayform solvates with water or common organic solvents. Such solvates areencompassed within the scope of the invention.

[0071] By “pharmaceutically acceptable derivative” is meant anypharmaceutically acceptable salt, hydrate or any other compound which,upon administration to the subject, is capable of providing (directly orindirectly) a compound of formula I or Ia or an antimicrobial orradioprotective active metabolite or residue thereof.

[0072] The term “pro-drug” is used herein in its broadest sense toinclude those compounds which are converted in vivo to compounds offormula I or Ia.

[0073] The term “tautomern” is used herein in its broadest sense toinclude compounds of formula I or Ia which are capable of existing in astate of equilibrium between two isomeric forms. Such compounds maydiffer in the bond connecting two atoms or groups and the position ofthese atoms or groups in the compound.

[0074] The term “isomer” is used herein in its broadest sense andincludes structural, geometric and stereo isomers. As the compound offormula I or Ia may have one or more chiral centres, it is capable ofexisting in enantiomeric forms.

[0075] The term “microbial infection” is used herein in its broadestsense and refers to any infection caused by a microorganism and includesbacterial infections, fungal infections, yeast infections and protozoalinfections.

[0076] The term “microorganism” includes any microscopic organism ortaxonomically related macroscopic organism within the categories algae,bacteria, fungi, yeast and protozoa or the like.

[0077] Bacterial infections include, but are not limited to, infectionscaused by Bacillus cereus, Bacillus anthracis, Clostridium botulinum,Clostridium difficile, Clostridium tetani, Clostridium perfringens,Corynebacteria diphtheriae, Enterococcus (Streptococcus D), Listeriamonocytogenes, Pneumoccoccal infections (Streptococcus pneumoniae),Staphylococcal infections and Streptococcal infections; Gram Negativebacteria including Bacteroides, Bordetella pertussis, Brucella,Campylobacter infections, enterohaemorrhagic Escherichia coli (EHEC/E.coli 0157: H7) enteroinvasive Escherichia coli (EIEC), enterotoxigenicEscherichia coli (ETEC), Haemophilus influenzae, Helicobacter pylori,Klebsiella pneumoniae, Legionella spp., Moraxella catarrhalis, Neisseriagonnorrhoeae, Neisseria meningitidis, Proteus spp., Pseudomonasaeruginosa, Salmonella spp., Shigella spp., Vibrio cholera and Yersinia;acid fast bacteria including Mycobacterium tuberculosis, Mycobacteriumavium-intracellulare, Myobacterium johnei, Mycobacterium leprae,atypical bacteria, Chlamydia, Mycoplasma, Rickettsia, Spirochetes,Treponema pallidum, Borrelia recurrentis, Borrelia burgdorfii andLeptospira icterohemorrhagiae and other miscellaneous bacteria,including Actinomyces and Nocardia.

[0078] Fungal infections include, but are not limited to, infectionscaused by Alternaria alternata, Aspergillus flavus, Aspergillusfumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillusversicolor, Blastomyces dermatiditis, Candida albicans, Candidadubliensis, Candida krusei, Candida parapsilosis, Candida tropicalis,Candida glabrata, Coccidioides immitis, Cryptococcus neoformans,Epidermophyton floccosum, Histoplasma capsulatum, Malassezia furfur,Microsporum canis, Mucor spp., Paracoccidioides brasiliensis,Penicillium marneffel, Pityrosporum ovale, Pneumocystis carinii,Sporothrix schenkii, Trichophyton rubrum, Trichophyton interdigitale,Trichosporon beigelii and Rhodotorula spp.

[0079] Yeast infections include, but are not limited to, infectionscaused by Brettanomyces clausenii, Brettanomyces custerii, Brettanomycesanomalous, Brettanomyces naardenensis, Candida himilis, Candidaintermedia, Candida saki, Candida solani, Candida tropicalis, Candidaversatilis, Candida bechii, Candida famata, Candida lipolytica, Candidastellata, Candida vini, Debaromyces hansenii, Dekkera intermedia,Dekkera bruxellensis, Geotrichium sandidum, Hansenula fabiani,Hanseniaspora uvarum, Hansenula anomala, Hanseniaspora guillermondiiHanseniaspora vinae, Kluyveromyces lactis, Kloekera apiculata,Kluveromyces marxianus, Kluyveromyces fragilis, Metschikowiapulcherrima, Pichia guilliermodii, Pichia orientalis, Pichia fermentans,Pichia memranefaciens, Rhodotorula Saccharomyces bayanus, Saccharomycescerevisiae, Saccharomyces dairiensis Saccharomyces exigus, Saccharomycesuinsporus, Saccharomyces uvarum, Saccharomyces oleaginosus,Saccharomyces boulardii, Saccharomycodies ludwigii, Schizosaccharomycespombe, Torulaspora delbruekii, Torulopsis stellata, Zygoaccharomycesbailli and Zygosaccharomyces rouxii.

[0080] Protozoal infections include, but are not limited to, infectionscaused by Leishmania, Toxoplasma, Plasmodia, Theileria, Anaplasma,Giardia, Trichomonas, Trypanosoma, Coccidia, and Babesia. Specificexamples include Trypanosoma cruzi, Eimeria tenella, Plasmodiumfalciparum, Plasmodium vivax or Plasmodium ovale.

[0081] Preferably, the microbial infection is an infection caused byeither a Gram Positive or a Gram negative bacterium, for example,Staphylococcus aureus, Enterococcus fecalis, Klebsiella pneumonia,Salmonella typhimurium or pseudotuberculosis, Acinetobacter, Pseudomonasaeruginosa, Clostridium perfringens, Clostridium difficile,Campylobacter jejuni or Bacteroides fragilis; a fungal or yeastinfection, for example, Trichophyton interdigitale; Aspergillusfumigatus or Candida albicans; or a protozoal infection, for examplePlasmodium falciparum or Trichomonas vaginalis.

[0082] Examples of microbial infections include bacterial or fungalwound infections, mucosal infections, enteric infections, septicconditions, pneumonia, trachoma, ornithosis, trichomoniasis, fungalinfections and salmonellosis, especially in veterinary practice. Thecompounds of the invention may also be used for the treatment ofresistant microbial species or in various fields where antiseptictreatment or disinfection of materials is required, for example, surfacedisinfection.

[0083] The term “subject” as used herein refers to any animal having adisease or condition which requires treatment with apharmaceutically-active agent. The subject may be a mammal, preferably ahuman, or may be a domestic or companion animal. While it isparticularly contemplated that the compounds of the invention aresuitable for use in medical treatment of humans, it is also applicableto veterinary treatment, including treatment of companion animals suchas dogs and cats, and domestic animals such as horses, ponies, donkeys,mules, llama, alpaca, pigs, cattle and sheep, or zoo animals such asprimates, felids, canids, bovids, and ungulates.

[0084] Suitable mammals include members of the orders Primates,Rodentia, Lagomorpha, Cetacea, Carnivora, Perissodactyla andArtiodactyla. Members of the orders Perissodactyla and Artiodactyla areparticularly preferred because of their similar biology and economicimportance.

[0085] For example, Artiodactyla comprises approximately 150 livingspecies distributed through nine families: pigs (Suidae), peccaries(Tayassuidae), hippopotamuses (Hippopotamidae), camels (Camelidae),chevrotains (Tragulidae), giraffes and okapi (Giraffidae), deer(Cervidae), pronghorn (Antilocapridae), and cattle, sheep, goats andantelope (Bovidae). Many of these animals are used as feed animals invarious countries. More importantly, many of the economically importantanimals such as goats, sheep, cattle and pigs have very similar biologyand share high degrees of genomic homology.

[0086] The order Perissodactyla comprises horses and donkeys, which areboth economically important and closely related. Indeed, it is wellknown that horses and donkeys interbreed.

[0087] As used herein, the term “effective amount” is meant an amount ofa compound of the present invention effective to yield a desiredantimicrobial or radioprotective activity.

[0088] The specific “effective amount” will, obviously, vary with suchfactors as the particular condition being treated, the physicalcondition of the subject, the type of subject being treated, theduration of the treatment, the nature of concurrent therapy (if any),and the specific formulations employed and the structure of the compoundor its derivatives.

[0089] The term “radiation damage” is used herein in its broadest senseand refers to damage resulting from exposure to a radiation source, suchas, ionising radiation. The term “ionising radiation” as used hereinrefers to photons having enough energy to ionise a bond, such as, α, βand γrays from radioactive nuclei and x-rays.

[0090] The term “cancer radiotherapy” is used herein in its broadestsense and include radiotherapy involving tumours which may be eitherbenign or malignant.

[0091] The primary application of the radioprotector of the presentinvention is in cancer radiotherapy. Many of the normal tissues whichare a problem in radiotherapy such as the skin, oral mucosa, oesophagealmucosa, rectal mucosa, vaginal mucosa and bladder epithelium can beprotected by the radioprotectors of the present invention.

[0092] Outside the context of cancer radiotherapy, the radioprotectorsof the present invention could be used prophylactly in high riskradiation situations.

[0093] The compounds of the present invention may additionally becombined with other medicaments to provide an operative combination. Itis intended to include any chemically compatible combination ofpharmaceutically-active agents, as long as the combination does noteliminate the activity of the compound of formula I or Ia. It will beappreciated that the compound of the invention and the other medicamentmay be administered separately, sequentially or simultaneously.

[0094] Other medicaments which may be used when treating microbialinfections include other anti-infective agents such as antibiotics.

[0095] When the compounds are used as radioprotectors the othermedicaments may include chemotherapeutic agents, for example,radiomimetic agents which are cytotoxic agents that damage DNA in such away that the lesions produced in DNA are similar to those resulting fromionising radiation.

[0096] Examples of radiomimetic agents which cause DNA strand breaksinclude bleomycin, doxorubicin, adriamycin, 5FU, neocarcinostatin,alkylating agents and other agents that produce DNA adducts. It isanticipated that the radioprotectors of the present invention willprotect DNA from damage by some of these agents, in the same way as theyprotect against the effects of ionising radiation. In clinicalapplications, it is unlikely that the radioprotector would beadministered systemically together with the chemotherapeutic agent,since this could compromise the action of this agent on the tumour.However, there are circumstances where topical application to problemtissues could be advantageous. For example, oral mucositis is problemside-effect for cytotoxic agents, such as, doxorubicin andadministration of the present radioprotector as a mouth-wash beforeadministration of the chemotherapeutic agent could ameliorate thisside-effect without compromising the action of this agent on a tumournot located in the oral cavity. Similarly, the gastrointestinal tractcould be protected by oral administration, the lungs by aerosolinhalation or the bladder by intravesical delivery, for example, via acatheter of the radioprotector. Hence a preferred method in accordancewith the present invention utilises the compound of formula I or Ia inconjunction with another medicament, such as, a radiomimetic agent.

[0097] The compounds of the invention may be conjugated to agents, forexample, via the interactive group, which will specifically deliver themto a desired tumour site. Suitable agents may include antibodies orproteins, growth factors, for example, haemopoietic growth factor whichwill enable preferential radioprotection of haemopoietic stem cells tooccur in the context of total body irradiation and bone marrowtransplantation.

[0098] There is also an ex vivo application of the conjugates of thecompounds of the invention in the context of bone marrowtransplantation. Bone marrow transplantation generally involvesobtaining and storing bone marrow samples from a subject in anticipationof a deterioration of their condition. A rather drastic form ofchemotherapy (i.e. a high dose) is then administered. This chemotherapyis such that it would normally be lethal due to the destruction ofnormal stem cells, but the subject is rescued by the administration oftheir own haemopoietic stem cells. The problem with this procedure isthat the initial sample of stem cells is likely to be contaminated withtumour cells and various procedures are use therefore to purge the bonemarrow preparations of the tumour cells. Radioprotectors conjugated to ahaemopoietic growth factor could be used in this context by being addedto a suspension of bone marrow cells. The suspension could then beirradiated in the expectation that the normal bone marrow cells, but notthe tumour cells, would be preferentially protected from thecell-killing effects of the radiation.

[0099] As used herein, a “pharmaceutical carrier” is a pharmaceuticallyacceptable solvent, suspending agent or vehicle for delivering thecompound of formula I or Ia to the subject. The carrier may be liquid orsolid and is selected with the planned manner of administration in mind.Each carrier must be pharmaceutically “acceptable” in the sense of beingcompatible with other ingredients of the composition and non injuriousto the subject.

[0100] The compound of formula I or Ia may be administered orally,topically, or parenterally in dosage unit formulations containingconventional non-toxic pharmaceutically acceptable carriers, adjuvants,and vehicles. The term parenteral as used herein includes subcutaneousinjections, aerosol for administration to lungs or nasal cavity,intravenous, intramuscular, intrathecal, intracranial, injection orinfusion techniques.

[0101] The present invention also provides suitable topical, oral andparenteral pharmaceutical formulations for use in the novel methods oftreatment of the present invention. The compounds of the presentinvention may be administered orally as tablets, aqueous or oilysuspensions, lozenges, troches, powders, granules, emulsions, capsules,syrups or elixirs. The composition for oral use may contain one or moreagents selected from the group of sweetening agents, flavouring agents,colouring agents and preserving agents in order to producepharmaceutically elegant and palatable preparations. Suitable sweetenersinclude sucrose, lactose, glucose, aspartame or saccharin. Suitabledisintegrating agents include corn starch, methylcellulose,polyvinylpyrrolidone, xanthan gum, bentonite, alginic acid or agar.Suitable flavouring agents include peppermint oil, oil of wintergreen,cherry, orange or raspberry flavouring.

[0102] Suitable preservatives include sodium benzoate, vitamin E,alphatocopherol, ascorbic acid, methyl paraben, propyl paraben or sodiumbisulphite. Suitable lubricants include magnesium stearate, stearicacid, sodium oleate, sodium chloride or talc. Suitable time delay agentsinclude glyceryl monostearate or glyceryl distearate. The tabletscontain the active ingredient in admixture with non-toxicpharmaceutically acceptable excipients which are suitable for themanufacture of tablets.

[0103] These excipients may be, for example, (1) inert diluents, such ascalcium carbonate, lactose, calcium phosphate or sodium phosphate; (2)granulating and disintegrating agents, such as corn starch or alginicacid; (3) binding agents, such as starch, gelatin or acacia; and (4)lubricating agents, such as magnesium stearate, stearic acid or talc.These tablets may be uncoated or coated by known techniques to delaydisintegration and absorption in the gastrointestinal tract and therebyprovide a sustained action over a longer period. For example, a timedelay material such as glyceryl monostearate or glyceryl distearate maybe employed. Coating may also be performed using techniques described inthe U.S. Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to form osmotictherapeutic tablets for control release.

[0104] The compound of formula I or Ia as well as thepharmaceutically-active agent useful in the method of the invention canbe administered, for in vivo application, parenterally by injection orby gradual perfusion over time independently or together. Administrationmay be intravenously, intraarterial, intraperitoneally, intramuscularly,subcutaneously, intracavity, transdermally or infusion by, for example,osmotic pump. For in vitro studies the agents may be added or dissolvedin an appropriate biologically acceptable solvent or buffer and added toa cell or tissue.

[0105] Preparations for parenteral administration include sterileaqueous or non-aqueous solutions, suspensions, and emulsions. Examplesof non-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's intravenousvehicles include fluid and nutrient replenishers, electrolytereplenishers (such as those based on Ringer's dextrose), and the like.Preservatives and other additives may also be present such as, forexample, antimicrobials, anti-oxidants, chelating agents, growth factorsand inert gases and the like.

[0106] Generally, the terms “treating”, “treatment” and the like areused herein to mean affecting a subject, tissue or cell to obtain adesired pharmacologic and/or physiologic effect. The effect may beprophylactic in terms of completely or partially preventing a disease orsign or symptom thereof, and/or may be therapeutic in terms of a partialor complete cure of a disease. “Treating” as used herein covers anytreatment of, or prevention of disease in a vertebrate, a mammal,particularly a human, and includes: (a) preventing the disease fromoccurring in a subject that may be predisposed to the disease, but hasnot yet been diagnosed as having it; (b) inhibiting the disease, i.e.,arresting its development; or (c) relieving or ameliorating the effectsof the disease, i.e., cause regression of the effects of the disease.

[0107] The invention includes various pharmaceutical compositions usefulfor ameliorating disease. The pharmaceutical compositions according toone embodiment of the invention are prepared by bringing a compound offormula I or Ia, analogues, derivatives or salts thereof, orcombinations of compound of formula I or Ia and one or morepharmaceutically-active agents into a form suitable for administrationto a subject using carriers, excipients and additives or auxiliaries.Frequently used carriers or auxiliaries include magnesium carbonate,titanium dioxide, lactose, mannitol and other sugars, talc, milkprotein, gelatin, starch, vitamins, cellulose and its derivatives,animal and vegetable oils, polyethylene glycols and solvents, such assterile water, alcohols, glycerol and polyhydric alcohols. Intravenousvehicles include fluid and nutrient replenishers. Preservatives includeantimicrobial, anti-oxidants, chelating agents and inert gases. Otherpharmaceutically acceptable carriers include aqueous solutions,non-toxic excipients, including salts, preservatives, buffers and thelike, as described, for instance, in Remington's PharmaceuticalSciences, 20th ed. Williams & Williams (2000), the British NationalFormulary, 43^(rd) edition (British Medical Association and RoyalPharmaceutical Society of Great Britain, 2000), the contents of whichare hereby incorporated by reference. The pH and exact concentration ofthe various components of the pharmaceutical composition are adjustedaccording to routine skills in the art. See Goodman and Gilman's ThePharmacological Basis for Therapeutics (7th ed., 1985).

[0108] The pharmaceutical compositions are preferably prepared andadministered in dose units. Solid dose units may be tablets, capsulesand suppositories. For treatment of a subject, depending on activity ofthe compound, manner of administration, nature and severity of thedisorder, age and body weight of the subject, different daily doses canbe used. Under certain circumstances, however, higher or lower dailydoses may be appropriate. The administration of the daily dose can becarried out both by single administration in the form of an individualdose unit or else several smaller dose units and also by multipleadministration of subdivided doses at specific intervals.

[0109] The pharmaceutical compositions according to the invention may beadministered locally or systemically in a therapeutically effectivedose. Amounts effective for this use will, of course, depend on theseverity of the disease and the weight and general state of the subject.

[0110] Typically, dosages used in vitro may provide useful guidance inthe amounts useful for in situ administration of the pharmaceuticalcomposition, and animal models may be used to determine effectivedosages for treatment of the microbial infections. Variousconsiderations are described, e.g., in Langer, Science, 249: 1527,(1990). Formulations for oral use may be in the form of hard gelatincapsules wherein the active ingredient is mixed with an inert soliddiluent, for example, calcium carbonate, calcium phosphate or kaolin.They may also be in the form of soft gelatin capsules wherein the activeingredient is mixed with water or an oil medium, such as peanut oil,liquid paraffin or olive oil.

[0111] Aqueous suspensions normally contain the active materials inadmixture with excipients suitable for the manufacture of aqueoussuspension. Such excipients may be (1) suspending agent such as sodiumcarboxymethyl cellulose, methyl cellulose, hydroxypropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;(2) dispersing or wetting agents which may be (a) naturally occurringphosphatide such as lecithin; (b) a condensation product of an alkyleneoxide with a fatty acid, for example, polyoxyethylene stearate; (c) acondensation product of ethylene oxide with a long chain aliphaticalcohol, for example, heptadecaethylenoxycetanol; (d) a condensationproduct of ethylene oxide with a partial ester derived from a fatty acidand hexitol such as polyoxyethylene sorbitol monooleate, or (e) acondensation product of ethylene oxide with a partial ester derived fromfatty acids and hexitol anhydrides, for example polyoxyethylene sorbitanmonooleate.

[0112] The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleagenous suspension. This suspension may beformulated according to known methods using those suitable dispersing orwetting agents and suspending agents which have been mentioned above.The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally-acceptable diluent orsolvent, for example, as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution, and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose, any bland fixed oil may be employedincluding synthetic mono-or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables.

[0113] The compound of formula I or Ia may also be administered in theform of liposome delivery systems, such as small unilamellar vesicles,large unilamellar vesicles, and multilamellar vesicles. Liposomes can beformed from a variety of phospholipids, such as cholesterol,stearylamine, or phosphatidylcholines.

[0114] The compound of formula I or Ia may also be presented for use inthe form of veterinary compositions, which may be prepared, for example,by methods that are conventional in the art. Examples of such veterinarycompositions include those adapted for:

[0115] (a) oral administration, external application, for exampledrenches (e.g. aqueous or non-aqueous solutions or suspensions); tabletsor boluses; powders, granules or pellets for admixture with feed stuffs;pastes for application to the tongue;

[0116] (b) parenteral administration for example by subcutaneous,intramuscular or intravenous injection, e.g. as a sterile solution orsuspension; or (when appropriate) by intramammary injection where asuspension or solution is introduced in the udder via the teat;

[0117] (c) topical applications, e.g. as a cream, ointment or sprayapplied to the skin; or

[0118] (d) intravaginally, e.g. as a pessary, cream or foam.

[0119] Dosage levels of the compound of formula I or Ia of the presentinvention may be of the order of up to about 1 g per kilogram bodyweight. The amount of active ingredient that may be combined with thecarrier materials to produce a single dosage will vary depending uponthe host treated and the particular mode of administration. For example,a formulation intended for oral administration to humans may contain upto about 1 g of an active compound with an appropriate and convenientamount of carrier material which may vary from about 5 to about 95percent of the total composition. Dosage unit forms will generallycontain between from about 5 mg to about 500 mg of active ingredient.

[0120] Optionally the compounds of the invention are administered in adivided dose schedule, such that there are at least two administrationsin total in the schedule. Administrations are given preferably at leastevery two hours for up to four hours or longer; for example the compoundmay be administered every hour or every half hour.

[0121] In one preferred embodiment, the divided-dose regimen comprises asecond administration of the compound of the invention after an intervalfrom the first administration sufficiently long that the level of activecompound in the blood has decreased to approximately from 5-30% of themaximum plasma level reached after the first administration, so as tomaintain an effective content of active agent in the blood. Optionallyone or more subsequent administrations may be given at a correspondinginterval from each preceding administration, preferably when the plasmalevel has decreased to approximately from 10-50% of theimmediately-preceding maximum.

[0122] It will be understood, however, that the specific dose level forany particular patient will depend upon a variety of factors includingthe activity of the specific compound employed, the age, body weight,general health, sex, diet, time of administration, route ofadministration, rate of excretion, drug combination and the severity ofthe particular disease undergoing therapy.

BRIEF DESCRIPTION OF THE FIGURES

[0123]FIG. 1 is a graph of Log. No. survivors vs time (hours) forCandida albicans in Example 9; and

[0124]FIG. 2 is a graph of % parasitised blood cells vs hours of culturein Example 26 in which T=Trophozoites, R=Rings and T/S=Trophozoites orSchizonts.

EXAMPLES

[0125] The invention will now be described in detail by way of referenceonly to the following non-limiting examples and drawings.

Example 1

[0126] General Synthesis Methods

[0127] Benzdioxols are described in the literature (Perekalkin, 1982a).The synthesis of benzoimidazole and benzoxazole may also be carried outusing standard condensation methods 1 and 2 (Perekalkin, 1966, 1982b) asshown below.

[0128] in which X, Y,

and R₁ to R₇ are as defined in formula I above.

[0129] In Method 2, equimolecular quantities of benzaldehyde andnitroalkane were mixed in an Erlenmayer flask and dissolved in equalvolume of alcohol. Fresh distillated ethylenediamine was added to theobtained solution in catalytical quantities (usually 1:10 in relation toaldehyde and nitroalkane) and was left in the dark at room temperaturefor several days (from 3 up to 10 days). During this time compoundcrystallised. After the cooling up to about 0° C., the crystals werefiltrated and washed with cold alcohol and then dried. When the yield issmall, the mother liquids can be joined together and evaporated inrotary evaporator. After cooling the additional quantity of impureproduct is obtained. The product was purified by dissolving in a minimalquantity of boiling alcohol. It was then treated with activated carbon,filtered hot and while the cooling was in progress, fine yellow needlescrystallised. The yield was about 80-85%, the compound beingchromatographically homogeneous.

[0130] The infrared spectra of the compounds obtained are in accordancewith those described in the literature (Hamlin and Weston, 1949;Knoevenagel and Walter, 1904; Burton and Duffield, 1949).

[0131] The compounds were soluble in organic solvents such as ethanol,acetone, benzene, methanol, acetonitrile, chloroform and DMSO, butshowed very poor solubility in water (0.1%). When an alcoholic solutionwas added to water, a stable colloidal mixture was formed.

Example 2

[0132] Method for Preparing Compound (1)(3,4-methylenedioxy-β-methyl-β-nitrostyrene)

[0133] Compound (1) was prepared using Method 1 described in Example 1above. The reaction scheme is shown below.

[0134] A mixture of 9.8 g of tetranitromethane (1 mole) and 10 cm³ ofacetone was cooled by ice and added dropwise to 8.1 g of distilledisosafrole (1 mole) and 4.8 g of pyridine (1.2 mole) dissolved in 20 cm³of acetone. The very first drops caused darkening of the reactionmixture and the liquid turned non-transparent and murky red when theentire portion of tetranitromethane was added. The smell oftetranitromethane disappeared quickly and in approximately 2 hours thedark red solution which had turned transparent was poured into 100 cm³of water in a stoppered bottle. The mixture was thoroughly shaken,covered with a layer of ether and a mixture of 6.7 cm³ of 33% solutionof caustic potassium (1.03 mole) and 50 cm³ of water was added in smallportions. The mixture was shaken after each addition and once the entireamount of alkali was added, the shaking was continued until the entiresalt of pyridine and nitroform, which is present as a dark red oil,disappeared. The water layer was then separated and again extracted withether. Combined ether extracts were first rinsed with water and thenwith water acidified with sulphuric acid and finally once again withpure water. After distillation of the ether in the vacuum, a sediment ofβ-nitroisosafrole was to be found in the form of yellow needles, whichwere re-crystallized from approximately 65 cm³ of alcohol. Compound (1)was obtained with a melting point of 98° C. and a yield of 7 g. Once thesolvent had evaporated, another 0.5 g of Compound (1) was obtained. Thetotal product amounted to 72.5% of the theoretical yield.

Example 3

[0135] Alternative Method for Preparing Compound (1)(3,4-methylenedioxy-β-methyl-β-nitrostyrene)

[0136] Compound (1) was prepared using Method 2 described in Example 1above. The reaction scheme is shown below.

[0137] 900 gm piperonal was dissolved in 1000 cc alcohol with constantshaking and 450 ml nitroethane was added slowly followed by 10 mlethyldiamine. After 17 hrs stirring, the mixture was placed in the darkat room temperature for 5-7 days. The resulting yellow crystals werefiltrated in a Buchner funnel until dried and then washed twice with 150ml alcohol. This yielded 1200 gm of Compound (1) with melting point of95° C. After further crystallization from ethanol, 1000 gm of lightyellow crystals were obtained with a melting point of 98° C. (approx 80%yield).

[0138] Molecular formula C₁₀H₉NO₄, molecular weight—207.05

[0139] Physical and Chemical Characteristics Form of state yellowcrystals Solubility profile soluble in ethanol, acetone, benzene,methanol, acetonitrile, chloroform, DMSO almost insoluble in waterMelting point 94-98° C. (when crystallized from 50% ethanol product had96-98° C.) pH (in 50% v/v ethanol) approximately neutral Specificrotation optically inactive but has 2 stereoisomers Stability begins todarken above 200° C. Purity MS indicates impurities of molecular weight303.4 & 331.4 to be the major impurities

[0140] IR Spectrum

[0141] 1. Aromatic ring—above 3000 wave number & associate aromatics1470-1630 region

[0142] 2. β-methylstyrene—additional groups over styrene

[0143] 1442 aliphatic —C— +900−1000 peaks

[0144] 3. Nitrogroup at low wave number e.g. 747, 673 and β-nitrostyrenehas 1520.

[0145]4. Aromatic Ether Group—1312 (1258) 1138, 1030 Nevertheless afingerprint of this compound is provided by the IR spectrum (q.v.). Thishas been done on the recrystallised material in order to reduce peaksdue to contaminants.

[0146] IR Spectrum

[0147] Impurities of molecules weight 303.4 & 331.4 Confirmation ofmolecular weight of main species 207.1

[0148] NMR Spectrum

[0149] Hydrogen NMR (200 MHz) shows: Aromatic ring with 3 remaining Hs,3 Hs as part of a CH₃, another attached to the side chain and 2 Hs aspart of another ring.

[0150] Carbon NMR (50 MHz) shows:

[0151] —CH₃, CH—, —CH₂ (as methylenedioxy)

[0152] Values of chemical shifts support the structure given and alikelihood of the E-stereoisomer rather than the Z-stereoisomer favouredby the synthesis used. A strong-withdrawing group (NO₂) is indicated.

[0153] UV/visible Spectrum

[0154] Recrystallised material has peaks (broad) at 250-270 mm and360-370 mm with high absorbance below 210 mm.

Example 4

[0155] Process for Preparing Compound (2)

[0156] Compound (2) was prepared using Method 2 described in Example 1above. The reaction scheme is shown below.

[0157] 3,4-methylenedioxybenzaldehyde was condensed with nitromethaneusing fresh distillated ethylenediamine NH2—CH₂—CH₂—NH₂ as a catalyst.The reaction was conducted in alcohol, darkness and at room temperaturefor 5 days. The resultant crystals were separated by filtration andwashed with cold alcohol. After being dried in air, the yield was 80%,m.p. −158-159° C. and after re-crystallization the m.p. was 162-163° C.Compound (2) was non-soluble in water, soluble in acetone, alcohol,acetic acid and in a majority of organic solvents.

Example 5

[0158] Antibacterial Activity

[0159] In the experiments described herein, museum strains of pathogensobtained from the museum of the Microbiology chair of the MilitaryMedical Academy (designated by the index “M”) and strains selected frompathological material (designated by the index “B”), taken from patientsand having gone through no more than three laboratory passages wereused. For each type of pathogen the corresponding optimal nutrient mediawas used. For the impregnation method, compounds (1) and (2) were addedto solid nutrient media at doses from 0.03% to 2.0%. An agar diffusionassay analogous to the standard method of determining sensitivity toantibiotics was used.

[0160] The agar diffusion assay was performed as follows.

[0161] Meat peptone agar was prepared and impregnated with the testcompounds at concentrations from 0.01 to 2.0%. The medium was pouredinto Petri dishes and allowed to set. Agar plugs of 10 mm diameter werecut out and placed on the surface of Petri dishes containing the samemedium immediately after they were inoculated with the microorganisms tobe investigated (at least 6 plugs per culture). After one day ofincubation at 37° C., the diameter of the zone of retardation of growthof the culture around the plugs was measured. The results were evaluatedin accordance with official standards of testing sensitivity toantibiotics; a diameter <20 mm corresponded to a stable culture, 21-28mm to moderate stability and >29 mm to sensitivity.

[0162] In parallel to this, the sensitivity of pathogens to 15antibiotics were tested according to the official protocol of theRussian Supervisory Authority for the Introduction of New MedicinalSubstances and Medical Technology (disc method).

[0163] Several pathogen types and strains were used to show the limitsof sensitivity of the strains and types to the test substances, in orderto evaluate their probable overall breadth of performance.

[0164] Table 1 below shows the results of experiments with Compound (1)at a concentration of 1.0%, at which it suppressed the propagation of5×10⁵-5×10⁷ organisms/mL, and comparative results of sensitivityexperiments using the following 15 antibiotics:

[0165] 1-penicillin,

[0166] 2-ampicillin,

[0167] 3-gentamycin,

[0168] 4-carbenicillin,

[0169] 5-kanamycin,

[0170] 6-lincomycin,

[0171] 7-levomicethin,

[0172] 8-oxacillin,

[0173] 9-polymixin,

[0174] 10-rifampicin,

[0175] 11-ristomycin,

[0176] 12-streptomycin,

[0177] 13-tetracycline,

[0178] 14-erythromycin,

[0179] 15-cephalosporin. TABLE 1 Suppression of Growth (+/−) CompoundAntibiotics Microorganism (1) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1.Conditional + − − − − − − − ± − − ± − ± − − pathogenic enterobacteriagram- negative aerobic bacilli Pseudomonas aeruginosa B-601 Escherichiacoli M-17 + + + + + + − + − + − − ± + + + Escherichia coliB-683 + + + + + + − + − + − − ± + + + Escherichia coliB-65 + + + + + + + + − + − + ± + + + Enterobacter aerogenes + − − − − −− − − + − − − − − − B-679 Enterobacter aerogenes + − − − − − − − − + − −− − − − B-687 Citrobacter diversus + − + + − + − + − ± − − ± ± − + B-6782. Pathogenic gram- + − ± + ± ± − + − ± ± − ± ± − ± negative bacteriaShigella flexneri M-2A6907 Shigella sonnei B-720 + − ± − ± ± − ± − ± ± −± ± − ± Salmonella typhimurium + − − − ± ± − − − ± ± − ± ± − ± M-727Salmonella paratyphi + − ± − ± ± − ± − − − − ± ± − ± M-16469Acinetobacter B-681 + − − − − − − − − − + − − − − − AcinetobacterB-676 + − − − − − − − − − + − − − − − Acinetobacter B-677 + − − − − − −− − − + ± − − − − Alkaligenes sp. B-689 + − − + − + − + − − − − ± − − −Alkaligenes sp. B-667 + − − − − − − − − + + − − − − − 3.Gram-negativenon- + − − − − − − − − − − − − − − − fermenting bacteria Yersiniapseudotuberculosis Klebsiella pneumoniae + − − − − − − − − − − − − − − −M-A21 Klebsiella pneumoniae + − − − − − − − − − − − − − − − M-248 4.Gram-nagative + − ± + − − − ± − − + + − + + + aerobic bacilliCorynebacterium diptheriae B-670 Bacillus sp. B-575 + − − ± − ± + + − −± ± ± + ± − 5. Gram-positive + − − − − − − − − − − − − − − − aerobiccocci (Neisseria) Neisseria meningitidis M-6231 Neisseria meningitidis +− − − − − − − − − − − − − − − M-A72 6. Gram-positive cocci + + − + + + −− + − + + + − − + (Staphylococci) Staphylococci aureus M-12159Staphylococci aureus + + − + + + − − − + + + + − − + M-209 Staphylococciaureus + − − + + + − − + − + + + − − + B-685 Staphylococci aureus + −− + − − − + − − − − ± + + + B-674 Staphylococci + + + + + − + − + − + +− − − + epidermidis D-513 Staphylococci + + + − + − − − + − + + − − − −epidermidis BK-30 7. Gram-positive cocci + + + − − − − − − − − + − − − −(Streptococci) γ-haemolytic streptococcus B-672 β-haemolytic + + + − + −− − − − + + − − − − streptococcus B-624

[0180] Table 2 below shows the results of experiments on sensitivity ofsome microorganisms to Compound (1) using the agar diffusion methoddescribed above. TABLE 2 Diameter of zone of Growth inhibition (mm)Concentration in disc of Compound (1) (%) Microorganism 2.0 1.0 0.1γ-haemolytic 35# 34# 32# streptococcus B-672 β-haemolytic 31# 32# 31#streptococcus B-624 Staphylococci aureus 39# 37# 35# B-685 Staphylococci38# 34# 32# epidermidis B-513 Shigella flexneri M- 36# 33# 31# 2A6907Shigella sonnei B- 39# 35# 30# 720 Salmonella 32# 30# 24# typhimuriumM-727 Salmonella paratyphi 36# 30# 28# M-16469 Acinetobacter B-681 38#35# 32# Alkaligenes sp. 40# 38# 36# B-689 Enterobacter 33# 30# 25#agglomerans B-679 Corynebacterium B- 40# 35# 31# 670 Bacillus sp. B-57538# 34# 29 

[0181] Table 3 shows the results of experiments of sensitivity ofpathogenic fungi to compound (1). TABLE 3 Number of microbes in thepresence of Compound (1) at Dosage of concentration (%) FungiInoculation Result 2.0 0.5 0.1 Candida 5 × 10⁴ Growth 0 0 0 albicansB-45 Retardation 5 × 10⁴ 5 × 10⁶ 5 × 10⁶ Candida 5 × 10⁴ Growth 0 0 0albicans M-3 Retardation 5 × 10⁶ 5 × 10⁶ 5 × 10⁶ Trichophyton 10² Growth0 0 0 Retardation 10⁷ 10⁷ 10⁷ Geotrichum 10⁵ Growth 0 0 0 M-158Retardation 10⁵ 10⁵ 10⁴ Torula 10⁵ Growth 0 0 0 histolytica Retardation10⁵ 10⁵ 10⁵ (Cryptococcus)

Example 6

[0182] Antimicrobial Tests

[0183] The antimicrobial activity of Compound (1) was tested, using theagar diffusion method.

[0184] Due to the very low solubility of the compounds, the studiescould not be carried out in the liquid phase. For this reason an initialmother impregnate, containing 0.5% of test compound, was prepared onmeat peptone agar. From this mother impregnate, regenerated to theliquid state in a water bath, serial double dilutions were prepared byaddition of the agar base. The dilutions thus obtained, containing thecompounds in concentrations of 0.5%, 0.25%, 0.12%, 0.06%, 0.03%, 0.015%were poured into Petri dishes, on which 6 bacterial and 2 fungal testcultures were inoculated.

[0185] The following test cultures were used:

[0186] 1) Staphylococcus aureus strain 674, isolated from a patient,sensitive to gentamycin, oxacillin, tetracycline,erythromycin andcephalothin, slightly sensitive to streptomycin.

[0187] 2) Enterococcus faecalis museum strain, sensitive to ampicillin,rifampicin, and streptomycin.

[0188] 3) Klebsiella pneumoniae strain 312, isolated from a patient,sensitive to gentamycin and polymyxin.

[0189] 4) Salmonella typhimurium museum strain 727, sensitive toampicillin, gentamycin, carbenicillin, canamicillin, polymyxin, andcephalothin.

[0190] 5) Acinetobacter strain 681, isolated from a patient, slightlysensitive to polymyxin.

[0191] 6) Pseudonomas aeruginosa strain 328, isolated from a patient,slightly sensitive to polymyxin.

[0192] 7) Trichophyton interdigitale

[0193] 8) Candida albicans.

[0194] Each of the 8 test cultures was sown on sterile meat peptone agarin a Petri dish, and then a standard plug of agar, impregnated with oneof the 9 compounds at a concentration of 0.5%, was placed on the agarsurface. The retardation zone was measured around the plug after 24 hand 48 h growth at 37° C. For the fungal cultures the results wereassessed after 7-10 days of incubation at 30° C.

[0195] The results of the action of the compound on the impregnated agarare summarised in Table 4. TABLE 4 Minimal % Compound (1) givingcomplete inhibition % inhibition of test culture by antibiotic S. aureus0.015 5.5 Enterococcus faecalis 0.015 3 Klebsiella pneumoniae 0.12 2Salmonella typhimurium 0.5 5 Acinotobacter 0.12 1 Pseudomonas — 1.5Trichophyton 0.25 — Candida 0.5 — Activity Index 0.4 —

[0196] Table 5 below shows the results of the agar diffusionexperiments. TABLE 5 Results of growth retardation by the compounds inthe diffusions in the agar experiment Size of zone of retardation Testculture of the growth by Compound (1) Staphylococcus aureus 6.5Enterococcus fecalis 6 Klebsiella pneumonia 5 Salmonella typhimurium 7Acinetobacteria 6.5 Pseudonomas 0 Average zone of retardation 5.17

[0197] In equal concentration Compound (1) inhibits the growth ofampicillin-resistant Staphylococcus and the growth of Enterococcus whichis sensitive to this antibiotic. Similar differences can be observedwith other pathogens and other preparations.

[0198] Anti TB Effects of Compound (1)

[0199] Anti TB effect was checked by standard method of serial doubledilutive in synthetic liquid medium (SOTON) with 10% normal equineserum. The solution was prepared in Tween 80.

[0200] Test culture was Mycobact tub. H.37RV sensitive to anti TBmedication.

[0201] Mycobacterial suspension (density 5×10⁷ cells/ml) was spread ontoa special liquid medium (Vischnevsky, B.I.)

[0202] Results were calculated for 10-14 days incubation at 37° C. MIC(totally inhibiting M. tuberculosis)

[0203] The results are shown in Table 6 below. TABLE 6 MinimalInhibitory Concentration (μg/ml) Compound (1) 6.25 Isoniazid 0.02-0.1 Rifampicin 0.01-0.02 Ethambutol 1.0-2.5 Streptomycin 0.5 

[0204] Only Compound (1) had MIC close to Ethambutol/Isoniazid MIC.

Example 7

[0205] Anti-protozoal Activity

[0206] The effect of the Compounds (1) and (2) on trichomonas was alsoinvestigated. Trichomonas vaginalis isolated from patients was used. Thetrichomonads were cultured at pH 5.8-6.5 and 37° C. in medium 199containing 5.0% native foetal calf serum, carbohydrates, and antibioticsto suppress the accompanying flora. Vaseline was applied to the surfaceof the medium in the culture tube. The experimental specimens containedthe test compounds at a concentration of 0.3%.

[0207] 7 specimens were investigated, containing motile forms of theparasite in a quantity of 5-8 cells in 1.0 cm³. In the control,parasites were cultured successfully over 3-4 passages (each passage 5-6days). In contrast, culture of motile forms in medium containing thetest compounds was unsuccessful in every case. After only one passage inthe presence of the test compounds, motile forms did not propagate.

Example 8

[0208] Anti-bacterial Activity in vivo

[0209] The therapeutic and prophylactic effect of the substances wasdetermined in experiments in vivo on mice, infected intraperitoneally orintranasally with Corynebacterium paratuberculosis.

[0210] Compounds (1) and (2) were administered intraperitoneally,intramuscularly and orally at a dose of <20% LD₅₀/0.2 at differentperiods from the day of infection, ie 2 and 1 days before the infection(schedules 2, 1), on the day of infection (schedule 0) and 1, 2, 3 etc.days after the infection (schedules +1, +2, +3, etc).

[0211] The daily mortality rate was measured, their cumulativevariations were calculated and based on this the results of theperformance of the preparation was determined by the formula

AI=[(B−A):B]×100

[0212] where

[0213] AI=activity index of the preparation (%),

[0214] A=cumulative mortality in the experimental group,

[0215] B=cumulative mortality in the control group.

[0216] The results are shown in Table 7 below and indicate thatCompounds (1) and (2) tested showed therapeutic and prophylacticactivity in mice infected with Corynebacterium paratuberculosis.

[0217] For intramuscular administration, the clinical prophylacticperformance in the form of reduced mortality of animals was 52.53%. Theclinical performance for salmonellosis varied within the limits of50.0-20.0%. For pseudotuberculosis the prophylactic performance was50.0%. TABLE 7 Order of activity of compounds as assessed by AIIncubation Cumu- period lative days mortality (test/ (test/ Pathogen andmethod of Schedule of introduction of control) control) Compoundinfection the preparation test contr. test contr. Activity index % 1Salmonella intraperitoneally −1, 0, +1, 0 0 47 99 +52, 53 +2, +3 200mg/kg intramuscularly 2 Salmonella intranasally +2, +3 1 0 15 30 +50, 00200 mg/kg intramuscularly

Example 9

[0218] Radiation Protective Activity

[0219] The radiation protective performance of the Compounds (1) and (2)investigated was tested on male white mice weighing 18-20 g.

[0220] Dosages: 2, 4, 6, 8, 10, 15, 20 Gr (1 hR=100 Röntgen).

[0221] Period of observation=25 days.

[0222] Method of reporting: dynamic mortality, calculation of cumulativemortality and factual changes of dosage (FCD).

[0223] Schedule of introduction: 50 mg/kg at 0.2 mL in mice.

[0224] Group 1-conntrol,

[0225] Group 2-2 and 1 days up to irradiation.

[0226] Table 8 shows the results of the experiments on radiationperformance of the compounds investigated. TABLE 8 Cumulative mortalityin groups % Dosage R (hR*) control prophylactic 2 2.9 0 4 3.6 0 6 13.13.8 8 21.1 9.5 10 43.8 13.3 15 66.7 45.5 20 94.1 83.3 FCD 1.39 + 1.48LD₅₀ 10.73 hR 15.88 hR

[0227] The results show that for all irradiation dosages prophylacticadministration of the test compounds considerably decreases themortality of irradiated animals in comparison with the control group.FCD (LD_(50 contr.))=1.39-1.43, which shows a high radiation protectiveeffect of the compounds indicated, the performances of which do notyield to the reported media.

Example 10

[0228] Treatment of Infected Wounds

[0229] Human volunteers suffering from skin wounds infected byStreptococcus and Staphylococcus were treated with 0.1% Compounds (1)and (2) in an ointment base of vaseline, sheep fat and sulfoxide.Application of 0.1% ointment for 3 days cleared the wound completely ofpus, with subsequent healing of the wound.

[0230] In 3 cases extensive damage to the skin caused by fungalinfection was treated; the type of the fungus was not identified. Thedamaged area was smeared twice per day with 0.1% ointment. The skin wascleared of fungal growth within a week.

Example 11

[0231] Pharmacokinetics

[0232] While the pharmacokinetics of the compounds of the invention havenot been investigated in detail, in experiments carried out in mice towhich Compounds (1) and (2) were administered intraperitonally andintragastrically, it has been established that the compound will remainin a biologically active concentration in the blood for longer than 24h.

Example 12

[0233] Toxicology

[0234] The average lethal dose for mice when administeredintragastrically was 1500 mg/kg body weight; when administeredintraperitonally the average lethal dose was 575 mg/kg body weight. Thusthe compounds of the invention have low toxicity.

Example 13

[0235] Antimicrobial Activity and Solubility of Compound (1)

[0236] Compound (1) is relatively insoluble in water but is soluble in10% DMSO at 1 mg/mL (0.1%), 10% ethanol at 2 mg/ml and 10% acetone at 2mg/mL.

[0237] The highest concentration testable is either 512 μg/mL at 5%solvent or 256 μg/mL at 2.5% solvent. It does not require specificchemical neutralization, dilution being sufficient to neutralizeresidual activity in microbicidal testing.

[0238] DMSO was selected as the solvent for testing because it has thelowest toxicity against test strains.

[0239] Compound (1) was at least 8 times more active against E. coliwhen formulated in ethanol, giving an MIC of 128 μg/mL (0.06% ETOH)compared to >512 μg/mL (2.5% DMSO). Ethanol is toxic to E. coli atconcentrations >2.5%.

Example 14

[0240] Antibacterial Activity of Compound (1)

[0241] NCCLS-USA Standard Method-Broth microdilution (or macrodilution)(Mueller-Hinton).

[0242] Inoculum 1-4×10⁴ cfu (or −4×10⁵ cfu) Ciprofloxacin test control.Chlorhexidine values added for disinfectant and antiseptic activitycomparison.

[0243] Minimum inhibitory concentration (MIC) and minimum bactericidalconcentration (MBC) as 3 log reduction (99.9% kill) at 35° C., 24 hours,aerobically (unless otherwise indicated). 48 h titres were notsignificantly different.

[0244] Results are shown in Table 9.

Summary of Results

[0245] Compound (1) is a relatively broad spectrum antibacterial agentwith bactericidal activity within an acceptable concentration range invitro for a representative selection of Gram positive and Gram negativebacteria. Compound (1) is broadly effective against aerobic Grampositive and Gram negative cocci and aerobic Gram positive rods ofclinical significance.

[0246] Infections with Resistant Gram Positive Cocci

[0247] Clinical isolates (multiple antibiotic resistance) of S. aureusand E. faecalis were as susceptible as the standard strains. Althoughnot active at the low concentrations cf current treatment drugs, theremay be a potential use for Compound (1) as a treatment for multiplyresistant staphylococci and enterococci not responding to the currentdrugs of choice.

[0248] Anaerobic Infections

[0249] Compound (1) is active against clinically significant anaerobes,Clostridium perfringens, Clostridium difficile and Bacteroides fragilis.C. difficile causes enterocolitis in hospitalized patients and iscurrently treated with vancomycin as the drug of choice. Induction ofresistance is a potential problem with vancomycin. There could be amarket for an oral drug for C. difficile enterocolitis as an alternativeto vancomycin.

[0250] Anaerobic infections are generally mixed infections of one ormore anaerobes with facultative bacteria, usually enteric Gram negativerods. The most common anaerobic pathogens are Clostridium difficile andBacteroides fragilis. Current treatment with metronidazole incombination with other antibacterial drugs is generally efficacious.Given Compound (1)'s broad spectrum against both aerobic and anaerobicbacteria this could possibly treat these infections.

[0251] Enteric Infections

[0252] Compound (1) is very active against Campylobacter jejuni.Campylobacter is currently the greatest cause of enteric infectionsworldwide and is often treated because of its severity in some patientsand the tendency for infection to predispose to development ofGuillain-Barre syndrome, a serious CNS disease.

[0253] The relative resistance of enteric Gram negative bacteria couldbe a function of solubility and ability of Compound (1) to penetratecells. The successful treatment of recalcitrant enteric infections andthe successful treatment of Salmonella infection in animals has beenshown.

[0254] Vulvo-vaginitis

[0255] Compound (1) is active against Neisseria gonorrhoeae.Vulvo-vaginitis is caused by Candida albicans, N. gonorrhoeae, Chlamydiatrachomatis and Trichomonas vaginalis (singly, not as co-infections).Compound (1) is active against two of these agents.

[0256] Formulating Compound (1) for greater solubility (and thereforeprobably greater absorption) could improve both its activity and itsdistribution in vivo. TABLE 9 MIC/MBC (μg/mL) for Compound (1),ciprofloxacin and chlorhexidine against a range of bacteria of clinicalsignificance Compound (1) Ciprofloxacin Chlorhexidine Bacterial strainMIC MMC MIC MMC MIC MMC Gram positive Staphylococcus aureus ATCC 2921316 16 0.25 0.25 2 8 S. aureus - clinical isolate 1 16 16 S. aureus -clinical isolate 2 16 16 Enterococcus faecalis ATCC 29212 32 32 0.5 0.5E. faecalis - clinical isolate 1 32 32 1 1 E. faecalis - clinicalisolate 2 32 32 Streptococcus pyogenes 16,8 16,8 Streptococcuspneumoniae ATCC49619 16 32 Bacillus subtilis RMIT 16 16 Corynebacteriumxerosis RMIT 32,16 32,16 Clostridium perfringens (48 h) 16 32Clostridium difficile 4 Gram negative Moraxella catarrhalis RMIT 32 32Neisseria gonorrhoeae WHO Strain VII 2 2 Haemophilus influenzae 0.1250.125 0.006 0.006 Bacteroides fragilis (48 h) 16 32 Campylobacter spp.(48 h) 128 128 Campylobacter jejuni RMIT FF3 (48 h) 2 2 Acinetobactercalcoaceticus RMIT 128 256 Proteus vulgaris RMIT 128 256 6 128 Proteusmirabilis 256 >512 Klebsiella oxytoca 128 256 Klebsiella pneumoniae512 >512 Salmonella Typhimurium 256 >512 Escherichia coli ATCC25922 >512 >512 0.02 0.02 2 4 Pseudomonas aeruginosa ATCC27853 >512 >512 0.25 0.25 32 64 Serratia marcescens RMIT >512 >512 16 32Enterobacter aerogenes 512 512

Example 15

[0257] Antifungal activity of Compound (1)

[0258] NCCLS-USA Broth macrodilution method (RPMI medium).

[0259] Inoculum ˜5×10⁴ hyphal fragments/mL (haemocytometer). Miconazolecontrol.

[0260] Minimum inhibitory concentration (MIC) and minimum fungicidalconcentration (MFC 2 log reduction −99% kill) at 30° C., aerobically, 2,7 and 10 days for yeasts and 4, 7 or 14 days for filamentous fungi.

[0261] Results are shown in Table 10.

Summary of Results

[0262] Compound (1) is fungicidal at relatively low concentrationsagainst a broad range of clinically significant yeasts and filamentousfungi (Table 10).

[0263] Dermatophyte Infections

[0264] Compound (1) shows good activity against 3 major causes of skin,hair and nail infections in humans and animals. Superficial fungalinfections are the most common fungal infections worldwide. Treatment isprolonged, over months (and years for nail infections). Superficialtreatments with antifungal lotions and creams is only partiallyeffective. Current treatments, although generally low cost, have poorefficacy and frequent relapse rates. Oral systemic agents (terbinafineand itraconazole) are preferred for superficial infections incompromised patients and nail infections.

[0265] Systemic Infections

[0266] Serious fungal infections in compromised patients have increasedworldwide in prevalence and severity. Fungal infections are generallylong term with a high therapeutic failure rate, frequent relapse anddevelopment of resistance by fungi. Candidiasis (Candida albicans) andaspergillosis (Aspergillus fumigatus) are the major fungal infections.Severe, invasive infections have a high mortality rate. Very feweffective drugs are available (cf antibacterial drugs). Long termtherapy makes safety and failure to induce resistance importantconsiderations. Amphotericin B is the main drug of choice for manyserious mycoses. It is fungicidal, with poor solubility and lowbioavailability and is limited by toxicity and delivery problems andhigh therapy failure. The azoles and triazoles are fungistatic drugs (egfluconazole and itraconazole) which have low toxicity, goodpharmacokinetic characteristics but are often ineffective due todevelopment of resistance on long term therapy.

[0267] Compound (1) has a broad spectrum, is fungicidal and has failedto induce resistance in C. albicans and A. fumigatus (see below). TABLE10 MIC/MFC (μg/mL) for Compound (1) and miconazole against clinicallysignificant fungi Compound (1) Miconazole Fungi MIC MMC MIC MMCDermatophytes (7 day) Trichophyton rubrum 1 256 1 64 Epidermophytonfloccosum 0.5 16 0.25 0.25 Microsporum gypseum 1 8 4 64 Yeasts (4 day)Candida albicans 8 8 4 8 Rhodotorula rubra 8 8 8 32 Filamentous fungi (7day) Fusarium graminearum 4 8 32 32 Rhizopus stolonifer 4 16 Aspergillusfumigatus 8 32 Penicillium chrysogenum 1 2

Example 16

[0268] Sporicidal Activity of Compound (1)

[0269]Bacillus subtilis Endospores

[0270]Aspergillus fumigatus Asexual Exospores

[0271] Compound (1) in water +Tween 20 was tested for sporicidalactivity up to 24 hours.

[0272] Compound (1) 512 μg/mL did not kill B. subtilis endospores in 24hours.

[0273] Compound (1) 512 μg/mL killed A. fumigatus exospores at 24 hoursbut not 6 hours.

[0274] Inhalation of Aspergillus spores is the major mechanism oftransmission. Activity against spores could be significant inprophylaxis of compromised individuals. Since the spore must germinateto infect, however, ultimately it is activity against vegetative formsof fungi that determine efficacy.

Example 17

[0275] Development of Resistance of Compound (1)

[0276] Bacterial and fungal strains were exposed to Compound (1) insub-inhibitory concentrations continuously for 12 weeks and monitoredfor a rise in MIC indicating development of resistance mechanisms. Aheavy and variable inoculum is used for weekly subculture so inhibitoryconcentrations each week vary. The standardized MIC is measured at thebeginning and end of exposure. A greater than 4-fold variation ofstandardized MIC is indicative of increased resistance, or a risingtrend in weekly MIC. The genera selected are known to develop resistancereadily to many antibiotics and to be a major clinical problem.

[0277] Bacterial species and C. albicans, known to develop resistance tomany current drugs, did not develop resistance to Compound (1) after 12weeks continuous exposure (Table 11). Strains were scanned for abnormalmicroscopic and macroscopic changes. Proteus vulgaris lost the abilityto swarm, indicating an effect on flagella. Other strains appearednormal. Tests are not yet complete for R. rubra and 3 moulds. Failure toinduce the development of resistance in these strains is a significantattribute of Compound (1). TABLE 11 Increase in MIC (μg/mL) of bacterialand fungal strains after 12 weeks continuous exposure to sub-inhibitoryconcentrations of Compound (1) in MHB Standard 7 day MIC μg/mL MIC μg/mLno rising trend Test strains Initial Final Range to Week 12Staphylococcus aureus 16 16 32-128 (MRSA clin isolate) Enterococcusfaecalis 32 32 64-128 (MR clin isolate) Klebsiella oxytoca 128 256256-512  Proteus vulgaris 128 128 64-256 Candida albicans 4 4 16-64Rhodotorula rubra 8 8-32 to Wk 5 Aspergillus fumigatus 8 8-32 to Wk 5Rhizopus stolonifer 4 4-32 to Wk 5 Fusarium graminearum 4 4-16 to Wk 5

Example 18

[0278] Antibacterial Activity of Compound (1) in the Presence of Blood

[0279] The activity of Compound (1) and ciprofloxacin was determined inthe presence of plasma and whole blood (horse), by macrodilution methodin Mueller Hinton broth to 48 h.

[0280] Compound (1) appeared to be relatively unaffected by the presenceof 10% plasma and to be more active in the presence of 5% whole blood.The slightly improved inhibitory activity of drugs in the presence ofblood sometimes occurs with antibacterial agents and is probably due tonatural antibacterial factors present in blood. Further increasing theconcentration of plasma and blood reduced the bactericidal activity ofCompound (1) against S. aureus as shown in Table 13. Ciprofloxacinshowed respectively a 4-fold and 2-fold decrease in activity against Saureus in the presence of 10% plasma and whole blood. TABLE 12 Activityof Compound (1) in the presence of human whole blood and plasma μg/mL —10% plasma 5% blood MIC MIC MMC MIC MMC S. aureus 16 16 256 16 256 B.subtilis 16 8 8 8 16 A. calcoaceticus 128 128 128 64 256 M. catarrhalis32 8 8 4 8

Example 19

[0281] Compound (1) Binding to Plasma Proteins

[0282] The MIC of Compound (1) was determined in increasingconcentrations of plasma. Compound (1) has been shown to bind to humanserum albumin and to agarose. Serum binding is significant in drugdistribution and bioavailability.

[0283] The MIC of Compound (1) is significantly increased withincreasing plasma concentrations.

[0284] Bactericidal activity is much more affected than inhibitoryactivity. Bioavailability of Compound (1) is significantly decreased inthe presence of plasma proteins (Table 13). Compound (1) is reversiblybound to proteins. TABLE 13 MIC/MMC (μg/mL) for Compound (1) in MHB inthe presence of increasing concentrations of plasma Streptococcus %plasma in S. aureus pyogenes MHB MIC MIC 0 4 4 1 4 4 2.5 8 8 5 8 8 10 1616 20 32 32 50 128 64 100 128 256

Example 20

[0285] Rate of Kill

[0286] Test strains were inoculated into Compound (1) solutions in waterand sampled immediately and at 1, 2, 4, 6 and 9 hours. Survivors wereestimated by viable counts on MHA (35° C., 48).

[0287] Measure of kill: reduction in viable count (log) expressed as logreduction factors.

[0288] (e.g., a I log reduction=90% kill, 2 log=99% kill, 3 log=99% killetc.)

[0289] Compound (1) showed rapid kill only against Candida albicans,with greater than a 99.999% reduction within 2 hours at 512 μg/mL andwithin 4 hours at 256 μg/mL as shown in FIG. 1.

[0290] Rate of kill was much slower against bacteria. The kill rate at512 μg/mL was 99.99% within 2 hours for B. subtilis and 9.99% within 9hours for S. aureus.

[0291] Ciprofloxacin was not tested. TABLE 14 Log reduction factors forCompound (1) over 6 or 9 hours Log reduction in viable count at time (h)Compound (1) μg/mL 512 256 128 Staphylococcus aureus   4 in 9 h 0.5 in 6h Enterococcus faecalis 0.6 at 9 h Bacillus subtilis   4 in 2 h 3.8 in 2h Klebsiella oxytoca   0   0 Proteus vulgaris   1 in 6 h 0.5 in 6 hAcinetobacter calcoaceticus   1 in 6 h   1 in 6 h Candida albicans 5.5at 2 h   6 in 4 h 2 in 6 h

Example 21

[0292] Dosing Range Test of Compound (1) in Rat

[0293] The aim of this example was to establish absorption and bloodlevels of Compound (1) in the rat after a single dose oraladministration.

[0294] Test Protocol

[0295] Sprague-Dawley rats (6 w/o, delivered 30 Jan, 2001) wereacclimatised for 6 days in the Animal Facility under standardizedenvironmental conditions (22° C. ±3° C., rel hum 30-70%, artificiallight, 12 h light/12 h dark). Rats were fed a conventional laboratorydiet with food and water ad lib and caged 5 rats per cage.

[0296] Test Substance

[0297] Compound (1) was prepared as an aqueous suspension in sterileLPW. At higher concentrations the suspension was sonicated to reduceparticle size sufficiently to pass through the gavage needle.

[0298] Compound (1) was tested at 1250, 1000, 500 and 100 mg/kg.

[0299] Test Method

[0300] Rats were randomly assigned to treatment groups, identified bynumbering on tails. Doses were tested sequentially from the lowest dose.Group A  100 mg/kg  5/2/01 B  500  8/2/01 (not fasted) C  500 12/2/01(fasted) D 1000 14/2/01 E 1250 21/2/01

[0301] Compound (1) suspensions and the water control were administeredat approx 100 mL/kgbw, in a single. One control and five treatment ratswere weighed immediately before each dose administration, the dosevolume calculated and the dose delivered by gavage (22 gauge stainlesssteel, smooth-balled end attached to a syringe).

[0302] Approximately 100-200 μL of blood (microfuge tube) was removedfrom the tail at 4 and 8 hours. Tails were prewarmed using a heat lampand snipped at the tip with a large scalpel. Blood was massaged into amicrofuge tube. Twenty four hour blood samples were not attemptedbecause of the difficulty of snipping scarred tails and the distresscaused to rats.

[0303] Blood was allowed to clot, centrifuged in a microfuge for 3minutes (speed 14) and the serum separated and stored at −20° C.

[0304] Animals were observed twice daily for 7 days and all observationsrecorded individually for each animal. Animals were not weighed afterthe initial weighing. Sacrifice and necropsy was performed at 7 days.

[0305] Animals were euthanized by carbon dioxide.

[0306] Gross pathology was recorded and samples of heart, lung, liver,kidney, stomach, spleen, duodenum and colon removed (10% formalin) forhistology.

Example 22

[0307] Blood Levels of Compound (1)

[0308] Bioassays

[0309] Bioassay for Compound (1) levels in blood was not possiblebecause of the interference due to strong binding of Compound (1) toblood proteins and to agar.

[0310] Agar Diffusion

[0311] An agar diffusion assay for Compound (1) was not possible becauseCompound (1) bound so strongly to agar that no zones of inhibition wereproduced at any concentration from 1 to 512 μg/mL with susceptiblestrains of S. aureus or Streptococcus pyogenes. Both well diffusion anddisk diffusion assays were attempted.

[0312] Broth Dilution

[0313] Dilution of serum in MHB and testing with a low inoculum of S.pyogenes (inhibited at 1 μg/mL of Compound (1) in MHB) was not possiblebecause strong binding to plasma at high concentrations caused asignificant prozone.

[0314] Assay by UV Spectroscopy

[0315] There was insufficient serum for assay of individual rat samples.

[0316] Samples for treatment groups and for controls were thus pooledand a mean level of Compound (1) for each treatment group determined.

[0317] Test Method

[0318] Compound (1) was extracted (×2) from serum by toluene andabsorbance measured at 370 nm (Hitachi U2000). A spiked control using100 μg/mL Compound (1) in 50% methanol/water (V/V) and untreatedcontrols were also assayed.

[0319] Blood Levels

[0320] Absorption of Compound (1) from the gastrointestinal tract isvery low, approximately 2% of the oral dose reaching the blood. Bloodlevels increased with dose level. Eight-hour levels were generallyhigher than 4-hour levels. The small difference between 4 and 8 hourlevels suggests a slow absorption. TABLE 15 Blood level of Compound (1)dose Sample time Compound (1) μg/mL (mg/kg) (hours) Mean  500 fed 4 8 817  500 fasted 4 12 8 14 1000 fasted 4 26 8 21 1250 4 + 8 h 27

Example 23

[0321] Antimicrobial Activity Spectrum of Compound (1)

[0322] Antifungal Activity

[0323] Filamentous Fungi

[0324] NCCLS-USA Broth Macrodilution method (RPMI medium) —draft.Inoculum 1-4×10⁵ cfu. Miconazole control. This test was used for initialactivity spectrum evaluations.

[0325] The MIC and MFC of filamentous fungi tested previously with theNCCLS-USA Broth Macrodilution Method were repeated using the newproposed standard microdilution method for testing fungi M38-PNCCLS-USA.

[0326] Results are of 2 or 3 replicates on different days.

[0327] Results were not significantly different from results obtainedwith the older method for all fungi previously tested. Amphotericin Bwas substituted for Miconazole as control for some tests.

[0328] Yeasts

[0329] NCCLS-M27-A Method for broth macro dilution antifungalsusceptibility testing of yeasts; approved standard.

[0330] M38-P microdilution method for filmentous fungi also used.

[0331] Minimum inhibitory concentration (MIC) and minimum fungicidalconcentration (MFC−2 log reduction−99% kill) at 35° C., 48 hours.Results of 2 or 3 replicates on different days for each method. Resultswere not significantly different for the two methods. M38P only reportedfor yeasts and filamentous fungi. TABLE 16 MIC/MFC (μg/mL) for Compound(1) and Amphotericin B against clinically significant fungi - M38 - Pmethod Amphotericin Compound (1) B Miconazole MIC MMC MIC MMC MIC MMCYeasts (24 h, 35° C.) Candida albicans 8 8 0.25₁ 0.25 C. guillermondii 22 0.03 0.06 1 1 RMIT 176 C. krusei RMIT 177 4 4 0.5₂ 0.5 2 2 C.parapsilosis RMIT 2 2 0.25₃ 0.25 0.5 0.5 178 C. tropicalis RMIT 4 4 0.250.5 1 2 181 C. glabrate RMIT 157 2 2 0.5 1 0.25 0.5 Cryptococcus 1 0.5 4neoformans Filamentous fungi (48 h, 35° C.) Aspergillus fumigatus 8 32 18 A. niger 8 16 2 2 A. flavus 16 32 8 8 Fusarium 4 8 4 8 graminearum F.chlamydosporum 8 8 2 2 Rhizopus stolonifer 4 16 >16 R. oryzae 64 64 4 4Rhizomucor pusillus 4 8 1 1 Paecilomyces variotii 1 2 1 1 Dematiaceousfungi Fonsecaea pedrosoi 2 2 8 16 Phialophora verrucosa 16 32 2 4Pseudoallescheria 2 4 4 16 boydii Dermatophytes Trichophyton rubrum 1256 Epidermophyton 0.5 16 floccosum Microsporum 1 8 gypseum

[0332] Bacteria

[0333] Methods

[0334] NCCLS-M7-A5 Standard Method—Broth microdilution (Mueller-Hinton).Inoculum 1-4×10⁴ cfu. Ciprofloxacin test control. Chlorhexidine andcetyl trimethyl ammonium bromide (CTAB) for disinfectant and antisepticactivity comparison.

[0335] NCCLS-M7-A5 Standard Method—macrodilution (MuellerHinton±specified enrichments) was also used. Minimum inhibitoryconcentration (MIC) and minimum bactericidal concentration (MBC) as 3log reduction (99.9% kill) at 35° C., 24 hours, aerobically. 48 h titreswere not significantly different and are not reported. Micro and macrodilution methods did not give significantly different MIC/MMC. TABLE 17μg/mL Compound (1) Ciprofloxacin Chlorhexidine CTAB Bacterial strain MICMMC MIC MMC MIC MMC MIC MMC Gram positive Staphylococcus aureus ATCC29213 16 16 0.25 0.25 2 8 16 32 S. aureus - clinical isolate 1 16 16 S.aureus - clinical isolate 2 16 16 Enterococcus faecalis ATCC 29212 32 320.5 0.5 E. faecalis - clinical isolate 1 32 32 E. faecalis - clinicalisolate 2 32 32 Streptococcus pyogenes RMIT 16 16 Streptococcuspneumoniae 2 2 Bacillus subtilis RMIT 16 16 Corynebacterium xerosis RMIT32 32 Gram negative Moraxella catarrhalis RMIT 32 32 Neisseriagonorrhoeae 2 2 Haemophilus influenzae 0.125 0.125 Acinetobactercalcoaceticus RMIT 128 256 Proteus vulgaris RMIT 128 128 6 128 128 256Proteus mirabilis 256 >512 Enterobacter aerogenes 512 512 Klebsiellaoxytoca 128 256 Klebsiella pneumoniae 512 >512 Escherichia coli ATCC25922 >512 >512 0.02 0.02 2 4 16 16 Pseudomonas aeruginosa ATCC27853 >512 >512 0.25 0.25 32 64 512 >512 Serratia marcescensRMIT >512 >512 16 32 128 128 Bacteroides fragilis 16 32

[0336] Campylobacter

[0337] Compound (1) was tested against a range of clinical strains ofCampylobacter spp. isolated from humans. TABLE 18 MIC/MMC (μg/mL) ofCompound (1) by NCCLS - M7 A5 macro dilution test, 42° C., 48 h,microaerophilic incubation MIC MMC Campylobacter jejuni 54/1-2 2 2 C.jejuni 541-3 2 2 C. coli 54/2 4 4 C. foetus 54/3 2 2 C. hyointestinalis54/4 2 2 C. sputorium 54/5 2 2 C. laniolis 54/6 2 2

[0338]Campylobacter is the most common cause of gastroenteritisinfection worldwide (bloody diarrhoea, abdominal pain, vomiting,headache, fever, lasting about 1 week). Sequelae are arthritis andGuillain-Barre syndrome(0.1%). It is acquired mainly from eatingpoultry. Incidence is about 2.5 million persons/year in USA. C. jejuniaccounts for 99% of cases. It can vary from sub-clinical to severe incompromised patients. It is usually untreated with only fluidreplacement or, if the disease is severe or threatening, withantibiotics (Erythromycin, tetracycline or fluoroquinolone). TABLE 19MIC/MFC (μg/mL) for Compound (1) (24 h, 35° C., O₂ - micro method)Bacteria MIC MMC Neisseria gonorrhoeae 2 2 Haemophilus influenzae 0.1250.125 Streptococcus pneumoniae 2 2

[0339] The above are significant human pathogens, all of which havesuccessful treatment regimens with antibiotics. N. gonorrhoeae is acause of vaginitis in women. Compound (1) is thus active at lowconcentrations against two causes, Candida albicans and N. gonorrhoeae.

[0340] Trichomonas Vaginalis

[0341] Method

[0342] The MIC of clinical isolate of Trichomonas vaginalis wasdetermined by macrobroth dilution in Diamond's complete medium, modifiedby Klass (Modified TYM) as described by Garcia, L. Cultures werecontained in 5 mL glass, screw-capped bottles without air-spaces.Volumes of 5 mL of log 2 dilutions, from 512 μg/mL Compound (1) in 5%DMSO to 0.25 μg/mL Compound (1) in 0.002% DMSO in modified TYM, wereinoculated with 0.5 mL volume of cells in log phase of growth giving afinal inoculum density of 1×10⁴ to 3×10⁴ cell/mL. Bottles were incubatedaerobically at 37° C. for 24 h before microscopic examination ofmotility. MIC was determined as the lowest concentration showing nomotility. Aliquots of 0.5 mL from all tubes showing no motility weresubcultured into further 5 mL volumes of modified TYM and incubatedaerobically at 37° C. for up to 5 days to confirm non-viability.

[0343] Tests were validated by growth controls in TYM, modified TYM with2.5% DMSO and modified TYM with 5% DMSO.

[0344] Tests were performed as 3 replicates on different days.

[0345] Results MIC MMC Trichomonas vaginalis 4 μg/mL 4 μg/mL

Example 24

[0346] Development of Resistance to Compound (1)

[0347] Selected resistant strains from the 12 week resistance testingwere retested simultaneously with parent strains using the newmicrodilution method. TABLE 20 Change in MIC (μg/mL) of fungal strainsafter 12 weeks continuous exposure to sub-inhibitory concentrations ofCompound (1) in MHB MIC μg/mL M38-P Test strains Initial FinalRhodotorula rubra 8 16 Aspergillus fumigatus 8 8 Rhizopus stolonifer 416 Fusarium graminearum 4 4

[0348] Fungi exhibit up to a four-fold difference in MIC on repeattesting. Significant increases in MIC are ≧8-fold.

[0349] There is no significant development of resistance by thefilamentous mould strains tested.

Example 25

[0350] Effect of Formulation in Ethanol on Activity

[0351] The effect of formulation in DMSO and ethanol on the MIC ofCompound (1) was compared for Candida albicans, Salmonella Typhimuriumand Escherichia coli using both the macrodilution and microdilutiontests.

[0352] Stock solutions in ethanol were allowed to stand for 48 h beforeuse to improve solubility. Compound (1) is not as soluble in ethanol asin DMSO. The concentration of ethanol and DMSO was kept constant at 2%and compared to a decreasing concentration of solvent on normal dilutionof the stock solution. There was no difference between the two testmethods. There was no difference in MIC between constant and decreasinglevels of DMSO. Only E. coli showed an enhanced susceptibility toethanol in the presence of a constant 2% ethanol. TABLE 21 Effect ofsolvent on MIC (μg/mL) of Compound (1) using microdilution DMSO EtOH(2%) MIC MBC MIC MBC Escherichia coli 512 >512 128 256 SalmonellaTyphimurium 512 >512 512 >512 Candida albicans 8 16 8 16

[0353] The synergistic effect of ethanol on E. coli was noted previouslywhen solvents were being tested for selection of an appropriate solventfor the drug. Ethanol has no enhancing effect on Staphylococcus,Salmonella or Candida. DMSO is a better solvent for the drug in in-vitrotests. Ethanol will be used for animal studies.

Example 26

[0354] Stability of Compound (1) Solutions on Storage

[0355] Stock solutions of Compound (1) at 512 μg/mL in water +5% DMSOwere stored at room temperature (RT˜18-21° C.), 4° C. and −20° C. for upto 12 weeks and the potency tested by measurement of MIC for Bacillussubtilis at 2-weekly intervals. TABLE 22 Stability of Compound (1) stocksolutions at 512 μg/mL tested by measurement of the MIC/MBC for Bacillussubtilis (24 h, 35° C.) at 2 weekly intervals RT₁ 4° C. −20° C.₂ Testtime MIC MBC MIC MBC MIC MBC 0 4 8 4 8 4 8 2 4 8 4 8 4 8 4 4 8 4 16 4 166 4 16 4 16 4 16 8 8 16 4 16 4 16 10 8 16 4 16 4 16 12 8 16 4 16 4 16

[0356]

[0357] Compound (1) is very stable, dilute solutions retaining potencyfor 12 weeks on storage at 4° C. and −20° C. Twofold loss of potency atroom temperature after 6 weeks is very low compared to working solutionsof antibiotics. It is also within the allowed variation range for MICmeasurements for bacteria (2-fold). Control antifungals were not tested.

Example 27

[0358] Effect of Compound (1) on Growth of the Human Malaria Parasite,Plasmodium Falciparum, in Human Red Blood Cells in Vitro

[0359] The aim of this example was to quantify the effect of Compound(1) on invasion and growth of the human malaria parasite Plasmodiumfalciparum in human red blood cells in vitro.

[0360] Methods

[0361] Malaria Parasites

[0362] 3D7 is a well characterised in vitro culture-adapted line of P.falciparum that was used for these experiments. The parasite undergoesrepeating cycles of growth and replication within human red blood cells.The duration of each complete cycle is 48 hours, beginning with youngring-stage parasites which mature through pigmented trophozoites duringthe first 24 hours of the cycle to segmented schizonts which burst torelease infectious merozoites which rapidly invade red blood cells.Newly invaded merozoites become ring forms, and the cycle continues.

[0363] Parasite Culture and Growth Inhibition Assays

[0364]P. falciparum parasites were maintained in synchronous in vitroculture in freshly collected human red blood cells, usingwell-established techniques. For invasion assays, red blood cellscontaining stage-synchronized mature, pigmented trophozoites werepurified and resuspended in fresh human red blood cells, so thatapproximately two in every 100 red blood cells was parasitised (2%parasitaemia). Fresh culture media was added to give a final red bloodcell concentration of 2×10⁸ red cells/ml.

[0365] Aliquots of the red blood cell suspension containing either thetest compound, the vehicle alone (in this case EtOH) or PBS (control)were incubated at 37° C. in an atmosphere of reduced oxygen tension (1%O₂, 3% CO₂, 96% N₂). Thin blood smears were made immediately (time=0)then subsequently after 24, 48 and 72 hours of culture. For each smear,parasitaemia and stage of parasite maturation was quantified bymicroscopic examination after staining with Giemsa at pH 7.2. Thisallowed invasion, parasite development and subsequent re-invasion to bequantified. At each sampling time point, the culture medium(±compound/vehicle) in all samples was completely replaced with freshmedium.

[0366] Compound (1) was tested as aqueous solutions of 100, 400 and 1000μg/ml each containing 10% EtOH. Stock solutions were stored at 4° C.until required. For the assay, each solution was further diluted 1:40 incomplete parasite culture medium (pH 7.2) to the desired workingconcentration (5, 10 and 25 μg/ml), then sterile filtered (0.22 μm)before being added to the parasitised red blood cell suspension. Stocksolutions were stored at 4° C. throughout the duration of the assay, anddiluted appropriately in parasite culture medium when required. Itshould be noted that at 1000 μg/ml, the compound was incompletelysoluble, even after warming to 37° C. and vigorous vortexing. Thus thetests performed at a putative concentration of 25 μg/ml, may in facthave been performed at a lower effective concentration.

[0367] Results

[0368] The effect of Compound (1) on parasite growth was tested at finalconcentrations of 5, 10 and 25 μg/ml. Results are presented graphicallyin FIG. 2. A concentration-dependent inhibitory effect on parasitegrowth and replication was detected at all concentrations of drugtested, being greatest at the highest concentration tested (25 μg/ml)after 72 hours of culture. EtOH alone, at a final concentration of 0.25%had no significant effect on parasite growth. All concentrations of thecompound tested showed no detectable adverse effect on red blood cellmorphology.

[0369] At 25 μg/mL no parasites were observed and at 10 μg/mL only veryfew were found, suggesting that the compound actually killed theparasites.

[0370] It will be apparent to the person skilled in the art that whilethe invention has been described in some detail for the purposes ofclarity and understanding, various modifications and alterations to theembodiments and methods described herein may be made without departingfrom the scope of the inventive concept disclosed in this specification.

[0371] References cited herein are listed on the following pages, andare incorporated herein by this reference.

[0372] REFERENCES

[0373] Burton, H., Duffield, G., J. Chem. Soc., 1949, 78

[0374] Denisenko P. P., Tarasenko A. A., Russian patent No. 2145215,“Substances having antimicrobial, antifungal, antiprotozoal activity”published 10^(th)February 2000

[0375] Foyer, G., Chemistry of nitro and nitroso groups, Moscow, 1973,Pt.2, pp.194-195

[0376] Garcia, L., Parasite culture: Trichomonas vaginalis, ClinicalMicrobiology Procedures Handbook, H. D. Isenberg (ed.), volume 2,American Society for Microbiology, Washington, USA, 7.9.3.1-7.9.3.6.

[0377] Hamlin, K., Weston, A., J. Am. Chem. Soc. 71, 2210 (1949)

[0378] Knoevenagel, E., Walter, L., Ber., 37, 4502 (1904)

[0379] Kuna P., Chemical radiation protection, Moscow, 1989, pp.25-28

[0380] Mashkovskiy M. D., Clinical agents, Pt.2, Moscow, 1986, p.189

[0381] Perekalkin V. V., Unlimited nitrocompounds, Leningrad, 1982,pp.55, 59, 61, 71, 73, 88, 89, 91, 95

[0382] Perekalkin V. V., Unlimited nitrocompounds, Leningrad, 1982, p.67

[0383] Perekalkin V. V., Unlimited nitrocompounds, Moscow, 1966, p.119

[0384] Vladimirov V. G. et al., Radiation protectors, structure andoperation, Kiev, 1989, p.139

1. A method of treatment and/or prophylaxis of a microbial infection,comprising the step of administering an effective amount of a compoundof formula I:

in which X and Y are either the same or different and selected from aheteroatom;

is a double or single bond depending on the heteroatoms X and Y; R₁ toR₅ are either the same or different and selected from hydrogen or anon-deleterious substituent; and R₆ and R₇ are either the same ordifferent and selected from hydrogen and a non-deleterious substituentor one of R₆ and R₇ are absent when there is a double bond present, orpharmaceutically acceptable salts or derivatives, pro-drugs, tautomersand/or isomers thereof.
 2. A method according to claim 1, in which themicrobial infection is a bacterial infection, fungal infection, yeastinfection, protozoal infection or viral infection.
 3. A method accordingto claim 1, in which the microbial infection is caused by a GramPositive bacterium.
 4. A method according to claim 1, in which themicrobial infection is caused by Staphylococcus aureus, Enterococcusfecalis, Klebsiella pneumonia, Salmonella typhimurium orpseudotuberculosis, Acinetobacteria, Pseudomonas aeruginosa, Clostridiumperfringens, Clostridium difficile, Campylobacter jejuni or Bacteroidesfragilis.
 5. A method according to claim 1, in which the microbialinfection is a fungal or yeast infection caused by Trichophytoninterdigitale, Aspergillus fumigatus or Candida albicans.
 6. A methodaccording to claim 1, in which the microbial infection is a protozoalinfection caused by Plasmodium falciparum or Trichomonas vaginalis.
 7. Amethod according to claim 1, in which the microbial infection is abacterial or fungal wound infection, mucosal infection, entericinfection, septic condition, pneumonia, trachoma, ornithosis,trichomoniasis or salmonellosis.
 8. A method according to claim 1, inwhich X and Y are either the same or different and selected from O andN.
 9. A method according to claim 8, in which X and Y are both O.
 10. Amethod according to claim 1, in which R₁ and R₂ are either the same ordifferent and selected from hydrogen, hydroxy, halogen or optionallysubstituted C₁₋₆ alkyl.
 11. A method according to claim 1, in which R₃to R₅ are either the same or different and selected from hydrogen,hydroxy, halogen, nitro, C₁₋₆ alkoxy or optionally substituted C₁₋₆alkyl.
 12. A method according to claim 10, in which the halogen ischlorine or bromine.
 13. A method according to claim 1, in which thecompound of the formula I is in the form of the E isomer.
 14. A methodaccording to claim 1, in which X, Y,

, R₆ and R₇ are as defined in claim 1; R₁ and R₂ are either the same ordifferent and selected from hydrogen, hydroxy, Cl, Br and C₁₋₄ alkyl;and R₃ to R₅ are either the same or different and selected fromhydrogen, hydroxy, Cl, Br, nitro, C₁₋₄ alkoxy or C₁₋₄ alkyl.
 15. Amethod according to claim 2, in which X and Y are O, R₁ is methyl and R₂and R₃ are hydrogen (3,4-methylenedioxy-β-methyl-β-nitrostyrene)

X and Y are O and R₁ to R₃ are hydrogen(3,4-methylenedioxy-β-nitrostyrene)

X is N, Y is NH, R₁ is methyl and R₂ and R₃ are hydrogen(benzimidazole-5-β-nitropropylene)

X is N, Y is NH, R₁ is hydrogen, R₂ is methyl and R₃ is absent (2-methylbenzimidazole-5-β-nitroethylene)

X is O, Y is N, R₁ and R₂ are hydrogen and R₃ is absent(benzoxazole-5-β-nitroethylene)

X is N, Y is O, R₁ and R₂ are methyl and R₃ is absent (2-methylbenzoxazole-5-β-nitropropylene)


16. A method according to claim 1, in which the microbial infection iscaused by a Gram Negative bacterium.
 17. A method according to claim 11,in which the halogen is chlorine or bromine.
 18. A method for protectinga subject from radiation damage which comprises administering aneffective amount of the compound of formula I as defined in claim 1 to asubject in need thereof.
 19. A method according to claim 18, in whichthe radiation is ionising radiation.
 20. A method of cancer radiotherapywhich comprises administering to a subject in need of such therapy aneffective amount of the compound of formula I as defined in claim 1 andsubjecting the locus of a tumour in the subject to a radiation source.21. A process according to claim 24 which is performed in the presenceof a catalyst.
 22. A compound of formula Ia:

in which X, Y,

and R₁ to R₇ are as defined in formula I above, with the provisos that:(i) when both X and Y are O and R₂ to R7 are hydrogen, then R₁ is nothydrogen, C₁₋₄ alkyl, CO₂Et,

or CH₂CO₂R₈ in which R₈ is C₁₋₁₂ alkyl or phenyl optionally substitutedby one or more halogen; (ii) when both X and Y are O, then R₁ to R₇ arenot hydrogen; (iii) when R₂, R₆ and R₇ are hydrogen, one of R₃, R₄ or R₅is methyl and X and Y are O, then R₁ is not methyl; (iv) when R₂, R₃,R₅, R₆ and R₇ are hydrogen, R₄ is OCH₃ and X and Y are O, then R₁ is notH, CH₃ or CH₂CH₃; (v) when R₁ to R₅ are H and X and Y are O, then atleast one of R₆ and R₇ is not methyl; (vi) when R₂, R₄, R₅, R₆ and R₇are H, R₃ is OCH₃ and X and Y are O, then R₁ is not CH₃; and (vii) whenR₁, R₂, R₃, R₅, R₆ and R₇ are H and X and Y are O, then R₄ is not OCH₃.23. A process for the preparation of the compound of formula Ia definedin claim 22 which comprises condensing a compound of formula II:

in which X, Y,

, R₃ to R7 are as defined in formula Ia in claim 22 with a compound offormula III: R₁R₂CHNO₂  III in which R₁ and R₂ are as defined in formulaIa in claim
 22. 24. A process for the preparation of the compound offormula Ia defined in claim 22 which comprises reacting a compound offormula IV:

in which X, Y,

, R₁ to R₇ are as defined in formula Ia in claim 22 with C(NO₃)₄.
 25. Aprocess according to claim 23 which is performed in the presence of acatalyst.
 26. A compound according to claim 22 together with apharmaceutically or veterinarily acceptable carrier.
 27. A compoundaccording to claim 26 in which the pharmaceutically or veterinarilyacceptable carrier is a topical, oral or parenteral carrier composition.28. A compound according to claim 26 in which the pharmaceutically orveterinarily acceptable carrier is an organic solvent.
 29. A compoundaccording to claim 28 in which the organic solvent is acetone, benzene,acetonitrile, DMSO or an alcohol.
 30. A method according to claim 1, inwhich the method of treatment and/or prophylaxis occurs in vivo.
 31. Amethod according to claim 1, in which the microbial infection is causedby a resistant microbial species.